Antibodies are preferred tools for biochemical and molecular biology research, diagnostics and medical applications due to their high affinity and specificity to the antigen and due to their relatively high stability in vitro and in vivo. Antibodies are made of two 25 heavy and two light chains, which contain the variable regions at their N-termini and which are linked by disulfide bridges. Single chain antibodies have been engineered by linking fragments of the variable heavy and light chain regions (scFv). Each variable domain contains three complementary determining regions (CDR) embedded in a framework. These CDRs are responsible for the interaction with the antigen. Each variable heavy and light region contains an intradomain disulfide bridge, which was reported to be critical for stability of the single chain antibody (Biocca et al., 1995; Derman et al., 1993).
The most commonly used technique to identify single chain antibodies which bind specific epitopes is by phage display and variations thereof (for review see Hoogenboom et al., 1998). This screening system has major advantages over conventional techniques like immunization or hybridoma technique, namely that it can uncover monoclonal single chain antibodies within a relatively short time.
Single chain antibodies expressed within the cell (e.g. cytoplasm or nucleus) are called intrabodies. Due to the reducing environment within the cell, disulfide bridges, believed to be critical for antibody stability, are not formed. Thus, it was initially believed that applications of intrabodies are not suitable. But several cases are described showing the feasibility of intrabodies (Beerli et al., 1994; Biocca et al., 1994; Duan et al., 1994; Gargano and Cattaneo, 1997; Greenman et al., 1996; Martineau et al., 1998; Mhashilkar et al., 1995; Tavladoraki et al., 1993). In these cases, intrabodies work by e.g. blocking the cytoplasmic antigen and therefore inhibiting its biological activity.
Up to now, intrabodies were most of the time derived from monoclonal antibodies which were first selected with classical techniques (e.g. phage display) and subsequently tested for their biological activity as intrabodies within the cell (Visintin et al., 1999). Although successful intrabodies are described (see above), it is today completely unpredictable whether such an intrabody is functional within the cell (for reviews see Cattaneo, 1998; Cattaneo and Biocca, 1999). The reasons are most probably the different environments: Phage display and other classical techniques are performed under oxidizing conditions, therefore disulfide bridges are formed, whereas intrabodies must function in reducing conditions. This reducing environment can lead to insufficient solubility of the intrabody and hence they form non-functional aggregates. The solubility of an intrabody can be modified by either changes in the framework (Knappik and Pluckthun, 1995) or the CDRs (Kipriyanov et al., 1997; Ulrich et al., 1995).
However, the hitherto known systems are limited with regard to their application to detect intracellular targets. Therefore, it is a growing need to have a reliable technology and system to screen directly for intrabodies specific for an antigen.
In WO 99/36569, Wittrup et al. describe a method to display proteins and scFv on the cell wall of yeast by using a yeast endogenous protein fragment derived from Aga2p for localization on the cell wall. Libraries of proteins and scFv can be screened interacting with other proteins. Other related systems are described in EP 0 407 259 (Boquet et al., 1991). These systems are comparable to the phage display screening where the protein or peptide library is also presented on the surface. However, these techniques cannot be used for intracellular screenings to identify intrabodies.
The patent document JP 11000174 (Kyoko et al., 1999) describes the use of yeast Pichia pastoris for high level expression and secretion of antibody Fab fragments. This yeast is famous for its high secretion level and is therefore preferably used for this application. The secreted antibody can be harvested by purification of the supernatant. Furthermore, in EP 0 590 067, WO92/22324, JP 060 30 778, U.S. Pat. No. 5,698,435, U.S. Pat. No. 5,595,889, JP 10313876 yeast is used for production of secreted proteins or antibodies. EP 0 698 097 and WO 94/25591 disclose application of the production and secretion of only the heavy chain or fragments thereof for further applications. JP 0 902 0798; JP 051 05700; and JP 050 97704 describe methods of yeast secretion to obtain hepatitis vaccine when administered to the human body or to organisms in general.
It is also already known from WO 99/28502 to use yeast for screenings of single chain antibodies. Said application discloses the use of a DNA construct library for a single chain monoclonal antibody fusion reagent. This scFv library (therein termed sFv library) is subsequently used for screenings. However, it has now been found that the stability and solubility of intrabodies can vary dramatically due to the use of a non specified framework. Furthermore, it could be shown that a direct correlation exists between the in vivo performance and the in vitro stability and solubility. Therefore, the use of mRNA derived libraries of different scFv fragments is limited in view of the possibility to identify CDR which have a high affinity to the antigen because, although the CDRs would in principle show the required high affinity to the antigen, the corresponding framework is not soluble enough and thus aggregates, making it impossible to select for this monoclonal scFv. Thus, there is still a need for improved antibodies, or intrabodies, respectively.
The growing applications of scFv directed against intracellular targets raise the need for reliable screening systems for intrabodies. Cytoplasmic targets of scFv are the most demanding application due to the instability of the scFv under reducing conditions and the unpredictability of the antibody stability. This stability and also solubility problem can be solved by using defined frameworks, optimized for intracellular application.